The main goal of Project 5 is to gain detailed information regarding the unique structural features of cross-linked hemoglobins (Hbs) that provide suitable properties as Hb-based oxygen carriers or blood substitutes. This information will help design "ideal" cross-linked Hbs with desired oxygen affinity, cooperativity, and stability which can function as acellular blood Synthetic blood substitutes are a pathogen-free alternative to available blood supplies. These blood substitutes will supplement or supplant blood reserves during crisis situations encountered in civilian and military settings, and will provide reagents with the required oxygen affinities to enhance sensitivity to radiation therapy. The production of acellular hemoglobin-based blood substitutes depends on the mimicry of hemoglobin (Hb) activity in the red blood cell. Thus, the design of these materials demands the incorporation of determinants for low oxygen affinity and high cooperativity, as well as the introduction of covalent cross-links to ensure tetramer stability. The structural characterization of selected Hb derivatives, using X-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy, will provide important insights into the relationship among structure, function, and dynamics. The continued study of selected mutant Hbs carrying alterations of the alpha2beta2 interface will provide considerable information about the origins of cooperativity and of the low oxygen affinity of the Hb molecule in the dexoy- (or T) state. The structural examination of covalently cross-linked Hbs will allow for an elaboration of the effects on tertiary and quaternary structures, and will identify the determinants responsible for modulating ligand affinity and cooperativity. Finally, the structural characterization of complexes between Hbs and novel, tight binding, potent allosteric effectors will further contribute to the understanding of cooperative ligand binding and the T-state oxygen affinity. The synthesis of this structural information will explain the synergism between specific mutants, cross-links and effector binding, and thus, will provide the structural framework necessary to design Hbs with the desired properties of an acellular blood substitute.